Set of genes for bladder cancer detection and use thereof

Abstract

Disclosed are a set of genes for bladder cancer detection and their use. The set of genes includes the following 32 genes: CA9 gene, CDK1 gene, CTSE gene, DMBT1 gene, ERBB2 gene, HOXA13 gene, IGF2 gene, CXCR2 gene, MAGEA3 gene, MDK gene, MMP1 gene, MMP12 gene, RBP2 gene, CCL18 gene, SNAI2 gene, VEGFA gene, MFAP5 gene, SGK2 gene, WFDC2 gene, POSTN gene, NPFFR2 gene, ANXA10 gene, CTAG2 gene, ZDHHC2 gene, KRT20 gene, PPP1R14D gene, FGD3 gene, AHNAK2 gene, SEMA3D gene, ZNF707 gene, LOC100652931 gene, and LINC00565 gene. After clinical validation, the kit provided by the present invention is used to detect bladder cancer with a high accuracy rate and objective interpretation of results. Meanwhile, as a non-invasive detection, the compliance of patients is greatly improved comparing with the existing cystoscopy, which has an important clinical significance for the early detection and postoperative monitoring of bladder cancer.

We claim: 
     
       1. A gene chip for a diagnosis, prognosis, or monitoring of bladder cancer, wherein the gene chip comprises:
 a solid phase carrier and a probe, wherein the probe is hybridized with sequences of 32 genes to be tested and/or complementary sequences of the 32 genes, wherein the probe is as set forth in SEQ ID NO: 65-SEQ ID NO: 96, respectively, wherein a set of genes comprises the following 32 genes: CA9 gene, CDK1 gene, CTSE gene, DMBT1 gene, ERBB2 gene, HOXA13 gene, IGF2 gene, CXCR2 gene, MAGEA3 gene, MDK gene, MMP1 gene, MMP12 gene, RBP2 gene, CCL18 gene, SNAI2 gene, VEGFA gene, MFAP5 gene, SGK2 gene, WFDC2 gene, POSTN gene, NPFFR2 gene, ANXA10 gene, CTAG2 gene, ZDHHC2 gene, KRT20 gene, PPP1R14D gene, FGD3 gene, AHNAK2 gene, SEMA3D gene, ZNF707 gene, LOC100652931 gene, and LINC00565 gene; 
 wherein the gene chip produces a result for bladder cancer detection. 
 
     
     
       2. A method of detecting bladder cancer using the gene chip according to  claim 1 , comprising the following steps:
 1) obtaining a urine sample for the bladder cancer detection; 
 2) isolating total RNA from the urine sample; 
 3) placing the total RNA in contact with the gene chip; 
 4) determining expression levels of the 32 genes in the urine sample; and 
 5) diagnosing a presence of the bladder cancer based on statistical analysis of the expression levels. 
 
     
     
       3. A method of preparing the gene chip according to  claim 1 , comprising using a data-driven analysis method to select gene combinations with a specificity to the bladder cancer as the set of genes, wherein the data-driven analysis method comprises the following steps:
 building a bladder cancer gene expression profile database, containing over 20,000 genes, 92 samples, and over 2 million data points; 
 correlating more than 20,000 human gene expression data in each of the 92 samples with clinical data of the 92 samples; 
 screening specific bladder cancer genes through a statistical analysis method T-test by analyzing a relevance of each of the specific bladder cancer genes to the bladder cancer; 
 extracting the specific bladder cancer genes with a highest correlation as signature genes, eventually obtaining the 32 genes for constructing a classifying model; and 
 establishing a statistical analysis model for the bladder cancer detection by using a Support Vector Machine, wherein for each sample to be tested, the statistical analysis model calculates a similarity score between a gene expression pattern of the sample to be tested and the bladder cancer in the bladder cancer gene expression profile database, and identifies whether the sample has the bladder cancer according to a principle of a highest similarity score. 
 
     
     
       4. The method according to  claim 2 , wherein the gene chip is arranged within a kit for the bladder cancer detection. 
     
     
       5. A kit for bladder cancer detection, wherein the kit comprises the gene chip according to  claim 1 , and biomarkers, wherein the biomarkers are the set of genes for the bladder cancer detection, wherein a result of the bladder cancer detection is bladder cancer positive or bladder cancer negative. 
     
     
       6. The kit according to  claim 5 , wherein the biomarkers are nucleic acids, oligonucleotide chains, or PCR primer sets. 
     
     
       7. The kit according to  claim 6 , wherein the biomarkers are PCR primer sets, wherein the PCR primer sets comprise:
 PCR primers for the CA9 gene, comprising: a forward primer as set forth in SEQ ID NO: 1, and a reverse primer as set forth in SEQ ID NO: 2; 
 PCR primers for the CDK1 gene, comprising: a forward primer as set forth in SEQ ID NO: 3, and a reverse primer as set forth in SEQ ID NO: 4; 
 PCR primers for the CTSE gene, comprising: a forward primer as set forth in SEQ ID NO: 5, and a reverse primer as set forth in SEQ ID NO: 6; 
 PCR primers for the DMBT1 gene, comprising: a forward primer as set forth in SEQ ID NO: 7, and a reverse primer as set forth in SEQ ID NO: 8; 
 PCR primers for the ERBB2 gene, comprising: a forward primer as set forth in SEQ ID NO: 9, and a reverse primer as set forth in SEQ ID NO: 10; 
 PCR primers for the HOXA13 gene, comprising: a forward primer as set forth in SEQ ID NO: 11, and a reverse primer as set forth in SEQ ID NO: 12; 
 PCR primers for the IGF2 gene, comprising: a forward primer as set forth in SEQ ID NO: 13, and a reverse primer as set forth in SEQ ID NO: 14; 
 PCR primers for the CXCR2 gene, comprising: a forward primer as set forth in SEQ ID NO: 15, and a reverse primer as set forth in SEQ ID NO: 16; 
 PCR primers for the MAGEA3 gene, comprising: a forward primer as set forth in SEQ ID NO: 17, and a reverse primer as set forth in SEQ ID NO: 18; 
 PCR primers for the MDK gene, comprising: a forward primer as set forth in SEQ ID NO: 19, and a reverse primer as set forth in SEQ ID NO: 20; 
 PCR primers for the MMP1 gene, comprising: a forward pruner as set forth in SEQ ID NO: 21, and a reverse primer as set forth in SEQ ID NO: 22; 
 PCR primers for the MMP12 gene, comprising: a forward primer as set forth in SEQ ID NO: 23, and a reverse primer as set forth in SEQ ID NO: 24; 
 PCR primers for the RBP2 gene, comprising: a forward primer as set forth in SEQ ID NO: 25, and a reverse primer as set forth in SEQ ID NO: 26; 
 PCR primers for the CCL18 gene, comprising: a forward primer as set forth in SEQ ID NO: 27, and a reverse primer as set forth in SEQ ID NO: 28; 
 PCR primers for the SNAI2 gene, comprising: a forward pruner as set forth in SEQ ID NO: 29, and a reverse primer as set forth in SEQ ID NO: 30; 
 PCR primers for the VEGFA gene, comprising: a forward primer as set forth in SEQ ID NO: 31, and a reverse primer as set forth in SEQ ID NO: 32; 
 PCR primers for the MFAP5 gene, comprising: a forward primer as set forth in SEQ ID NO: 33, and a reverse primer as set forth in SEQ ID NO: 34; 
 PCR primers for the SGK2 gene, comprising: a forward primer as set forth in SEQ ID NO: 35, and a reverse primer as set forth in SEQ ID NO: 36; 
 PCR primers for the WFDC2 gene, comprising: a forward primer as set forth in SEQ ID NO: 37, and a reverse primer as set forth in SEQ ID NO: 38; 
 PCR primers for the POSTN gene, comprising: a forward primer as set forth in SEQ ID NO: 39, and a reverse primer as set forth in SEQ ID NO: 40; 
 PCR primers for the NPFFR2 gene, comprising: a forward primer as set forth in SEQ ID NO: 41, and a reverse primer as set forth in SEQ ID NO: 42; 
 PCR primers for the ANXA10 gene, comprising: a forward primer as set forth in SEQ ID NO: 43, and a reverse primer as set forth in SEQ ID NO: 44; 
 PCR primers for the CTAG2 gene, comprising: a forward primer as set forth in SEQ ID NO: 45, and a reverse primer as set forth in SEQ ID NO: 46; 
 PCR primers for the ZDHHC2 gene, comprising: a forward primer as set forth in SEQ ID NO: 47, and a reverse primer as set forth in SEQ ID NO: 48; 
 PCR primers for the KRT20 gene, comprising: a forward primer as set forth in SEQ ID NO: 49, and a reverse primer as set forth in SEQ ID NO: 50; 
 PCR primers for the PPP1R14D gene, comprising: a forward primer as set forth in SEQ ID NO: 51, and a reverse primer as set forth in SEQ ID NO: 52; 
 PCR primers for the FGD3 gene, comprising: a forward primer as set forth in SEQ ID NO: 53, and a reverse primer as set forth in SEQ ID NO: 54; 
 PCR primers for the AHNAK2 gene, comprising: a forward primer as set forth in SEQ ID NO: 55, and a reverse primer as set forth in SEQ ID NO: 56; 
 PCR primers for the SEMA3D gene, comprising: a forward primer as set forth in SEQ ID NO: 57, and a reverse primer as set forth in SEQ ID NO: 58; 
 PCR primers for the ZNF707 gene, comprising: a forward primer as set forth in SEQ ID NO: 59, and a reverse primer as set forth in SEQ ID NO: 60; 
 PCR primers for the LOC100652931 gene, comprising: a forward primer as set forth in SEQ ID NO: 61, and a reverse primer as set forth in SEQ ID NO: 62; 
 PCR primers for the LINC00565 gene, comprising: a forward primer as set forth in SEQ ID NO: 63, and a reverse primer as set forth in SEQ ID NO: 64. 
 
     
     
       8. A method of detecting bladder cancer using the kit according to  claim 5 , comprising:
 1) obtaining a urine sample for the bladder cancer detection; 
 2) isolating total RNA from the urine sample; 
 3) placing the total RNA in contact with the biomarkers in the kit; 
 4) determining expression levels of the 32 genes in the urine sample; and 
 5) based on statistical analysis of the expression levels, performing a diagnosis, prognosis, or monitoring of the bladder cancer. 
 
     
     
       9. The method according to  claim 2 , wherein a result of the bladder cancer detection is bladder cancer positive or bladder cancer negative. 
     
     
       10. The method according to  claim 2 , wherein step 2 comprises the following steps: centrifuging desquamated cells in the urine sample, and then extracting the total RNA by a purification column extraction method using a purification column as a liquid elution medium. 
     
     
       11. The method according to  claim 3 , wherein the gene chip is arranged within a kit for the bladder cancer detection. 
     
     
       12. The method according to  claim 8 , wherein step 2 comprises the following steps: centrifuging desquamated cells in the urine sample, and then extracting the total RNA by a purification column extraction method using a purification column as a liquid elution medium. 
     
     
       13. A method of preparing the kit according to  claim 5 , comprising using a data-driven analysis method to select gene combinations with a specificity to the bladder cancer as the set of genes, wherein the data-driven analysis method comprises the following steps:
 building a bladder cancer gene expression profile database, containing over 20,000 genes, 92 samples, and over 2 million data points; 
 correlating more than 20,000 human gene expression data in each of the 92 samples with clinical data of the 92 samples; 
 screening specific bladder cancer genes through a statistical analysis method T-test by analyzing a relevance of each of the specific bladder cancer genes to the bladder cancer, 
 extracting the specific bladder cancer genes with a highest correlation as signature genes, eventually obtaining the 32 genes for constructing a classifying model; and 
 establishing a statistical analysis model for the bladder cancer detection by using a Support Vector Machine, wherein for each sample to be tested, the statistical analysis model calculates a similarity score between a gene expression pattern of the sample to be tested and the bladder cancer in the bladder cancer gene expression profile database, and identifies whether the sample has the bladder cancer according to a principle of a highest similarity score. 
 
     
     
       14. The method according to  claim 8 , wherein the biomarkers are nucleic acids, oligonucleotide chains, or PCR primer sets. 
     
     
       15. The kit according to  claim 14 , wherein the biomarkers are PCR primer sets, wherein the PCR primer sets comprise:
 PCR primers for the CA9 gene, comprising: a forward primer as set forth in SEQ ID NO: 1, and a reverse primer as set forth in SEQ ID NO: 2; 
 PCR primers for the CDK1 gene, comprising: a forward primer as set forth in SEQ ID NO: 3, and a reverse primer as set forth in SEQ ID NO: 4; 
 PCR primers for the CTSE gene, comprising: a forward primer as set forth in SEQ ID NO: 5, and a reverse primer as set forth in SEQ ID NO: 6; 
 PCR primers for the DMBT1 gene, comprising: a forward primer as set forth in SEQ ID NO: 7, and a reverse primer as set forth in SEQ ID NO: 8; 
 PCR primers for the ERBB2 gene, comprising: a forward primer as set forth in SEQ ID NO: 9, and a reverse primer as set forth in SEQ ID NO: 10; 
 PCR primers for the HOXA13 gene, comprising: a forward primer as set forth in SEQ ID NO: 11, and a reverse primer as set forth in SEQ ID NO: 12; 
 PCR primers for the IGF2 gene, comprising: a forward primer as set forth in SEQ ID NO: 13, and a reverse primer as set forth in SEQ ID NO: 14; 
 PCR primers for the CXCR2 gene, comprising: a forward primer as set forth in SEQ ID NO: 15, and a reverse primer as set forth in SEQ ID NO: 16; 
 PCR primers for the MAGEA3 gene, comprising: a forward primer as set forth in SEQ ID NO: 17, and a reverse primer as set forth in SEQ ID NO: 18; 
 PCR primers for the MDK gene, comprising: a forward primer as set forth in SEQ ID NO: 19, and a reverse primer as set forth in SEQ ID NO: 20; 
 PCR primers for the MMP1 gene, comprising: a forward pruner as set forth in SEQ ID NO: 21, and a reverse primer as set forth in SEQ ID NO: 22; 
 PCR primers for the MMP12 gene, comprising: a forward primer as set forth in SEQ ID NO: 23, and a reverse primer as set forth in SEQ ID NO: 24; 
 PCR primers for the RBP2 gene, comprising: a forward primer as set forth in SEQ ID NO: 25, and a reverse primer as set forth in SEQ ID NO: 26; 
 PCR primers for the CCL18 gene, comprising: a forward primer as set forth in SEQ ID NO: 27, and a reverse primer as set forth in SEQ ID NO: 28; 
 PCR primers for the SNAI2 gene, comprising: a forward pruner as set forth in SEQ ID NO: 29, and a reverse primer as set forth in SEQ ID NO: 30; 
 PCR primers for the VEGFA gene, comprising: a forward primer as set forth in SEQ ID NO: 31, and a reverse primer as set forth in SEQ ID NO: 32, 
 PCR primers for the MFAP5 gene, comprising: a forward primer as set forth in SEQ ID NO: 33, and a reverse primer as set forth in SEQ ID NO: 34; 
 PCR primers for the SGK2 gene, comprising: a forward primer as set forth in SEQ ID NO: 35, and a reverse primer as set forth in SEQ ID NO: 36; 
 PCR primers for the WFDC2 gene, comprising: a forward primer as set forth in SEQ ID NO: 37, and a reverse primer as set forth in SEQ ID NO: 38; 
 PCR primers for the POSTN gene, comprising: a forward primer as set forth in SEQ ID NO: 39, and a reverse primer as set forth in SEQ ID NO: 40; 
 PCR primers for the NPFFR2 gene, comprising: a forward primer as set forth in SEQ ID NO: 41, and a reverse primer as set forth in SEQ ID NO: 42; 
 PCR primers for the ANXA10 gene, comprising: a forward primer as set forth in SEQ ID NO: 43, and a reverse primer as set forth in SEQ ID NO: 44; 
 PCR primers for the CTAG2 gene, comprising: a forward primer as set forth in SEQ ID NO: 45, and a reverse primer as set forth in SEQ ID NO: 46; 
 PCR primers for the ZDHHC2 gene, comprising: a forward primer as set forth in SEQ ID NO: 47, and a reverse primer as set forth in SEQ ID NO: 48; 
 PCR primers for the KRT20 gene, comprising: a forward primer as set forth in SEQ ID NO: 49, and a reverse primer as set forth in SEQ ID NO: 50; 
 PCR primers for the PPP1R14D gene, comprising: a forward primer as set forth in SEQ ID NO: 51, and a reverse primer as set forth in SEQ ID NO: 52; 
 PCR primers for the FGD3 gene, comprising: a forward primer as set forth in SEQ ID NO: 53, and a reverse primer as set forth in SEQ ID NO: 54; 
 PCR primers for the AHNAK2 gene, comprising: a forward primer as set forth in SEQ ID NO: 55, and a reverse primer as set forth in SEQ ID NO: 56; 
 PCR primers for the SEMA3D gene, comprising: a forward primer as set forth in SEQ ID NO: 57, and a reverse primer as set forth in SEQ ID NO: 58; 
 PCR primers for the ZNF707 gene, comprising: a forward primer as set forth in SEQ ID NO: 59, and a reverse primer as set forth in SEQ ID NO: 60; 
 PCR primers for the LOC100652931 gene, comprising: a forward primer as set forth in SEQ ID NO: 61, and a reverse primer as set forth in SEQ ID NO: 62; 
 PCR primers for the LINC00565 gene, comprising: a forward primer as set forth in SEQ ID NO: 63, and a reverse primer as set forth in SEQ ID NO: 64.